Adsorbent For Lymphocyte Proliferation Inhibitor and Treating Method

ABSTRACT

The invention has for its object to provide a porous material capable of relieving the lymphocyte proliferation inhibition in lymphocyte culture and improving the proliferative nature of lymphocytes as well as a method for proliferating lymphocytes and a method for producing lymphocytes each of which utilizes such porous material. 
     The invention relates to a porous material for body fluid treatment for promoting lymphocyte proliferation in lymphocyte culture which contains a high-molecular compound having an angle of contact with water within the range of 40° to 98 °; and a porous material for body fluid treatment which comprises activated carbon. It also relates to a treatment device wherein the porous material is used; a method for proliferating lymphocytes; a method for producing mammalian lymphocytes; a method for producing a pharmaceutical composition; an additive body fluid to be added to a culture medium on the occasion of lymphocyte culture; a method for treating a disease against which a therapeutic effect is produced by returning extracorporeally activated mammalian lymphocytes into the body; and the use of activated carbon or a high-molecular compound having an angle of contact with water within the range of 40° to 98° in manufacturing porous materials for body fluid treatment for promoting the lymphocyte proliferation in lymphocyte culture.

TECHNICAL FIELD

The present invention relates to a porous material for overcoming thelymphocyte proliferation inhibition in lymphocyte culture in activatedautologous lymphocyte therapy, among others, which comprises takingimmunocompetent cells (lymphocytes in particular) in blood out of thebody, culturing them for stimulation/activation and for proliferationand again returning them into the body to thereby preventing the advanceof cancer, an infectious disease or an immune disease, and to a devicefor lymphocyte proliferation wherein the porous material mentioned aboveis utilized. Furthermore, it relates to a method for preparing a bodyfluid with a reduced lymphocyte proliferation inhibitor concentration aswell as to a method for proliferating lymphocytes using a medium withsuch body fluid added thereto.

BACKGROUND ART

In recent years, attention has been focused on activated autologouslymphocyte therapy, which comprises taking immunocompetent cells(lymphocytes in particular) in blood out of the body, culturing them forstimulation/activation and for proliferation and again returning theminto the body to thereby prevent the advance of cancer. This techniqueproduces little side effects and makes it possible to maintain thequality of life (QOL) at high levels even during treatment and,therefore, is becoming more and more popular in the field of cancertreatment as a fourth choice of cancer therapy next to the three majorcancer treatment methods, namely surgical therapy, radiotherapy andchemotherapy. The technique is already in actual use as one oftailor-made highly advanced medical treatment methods in universityhospitals, cancer centers and specialized clinics and, expectedly, itwill be used still more widely. This technique generally comprisescollecting a portion of the blood of a patient, separating a lymphocytefraction by density gradient centrifugation, adding the autologousplasma to a medium for exclusive use and cultivating the lymphocytes.Generally, the number of lymphocytes arrives at about 100 times thenumber of lymphocytes in the primary culture in a week. It has becomeknown, however, that there are some such cancer patients thatlymphocytes derived therefrom can hardly proliferate in the presence ofautologous plasma but can proliferate in the presence of the plasmaderived from another person (resulting from blood donation). Cancercells produce cellular immunity inhibiting factors, for example suchcytokines as transforming growth factor beta (hereinafter abbreviated asTGF-β), interleukin 4 (hereinafter abbreviated as IL4), interleukin 6(hereinafter abbreviated as IL6) and interleukin 10 (hereinafterabbreviated as IL10) as well as prostaglandin E2 (hereinafterabbreviated as PGE2). Thus, the possibility is suggested that suchfactors might inhibit the proliferation of lymphocytes. However, theconcentrations of these factors in the blood of cancer patients arealmost the same as those in persons in normal health, although theirlocal concentrations in cancer foci are high. Further, when such factorscommercially available as reagents are dissolved in plasma at highconcentrations and the influences thereof on lymphocyte proliferationare examined, little inhibition is observed. Such and other findingssuggest that there is an unknown mechanism other than the involvement ofsuch factors.

As a matter of fact, an adsorbent for adsorptively removingimmunosuppressive acidic proteins (IAPs) (Patent Document 1), anadsorbent for adsorptively removing interleukins in body fluids (PatentDocument 2) and an adsorbent capable of adsorbing TGF-β in body fluids(Patent Document 3), among others, have so far been disclosed. However,all of them are limited in scope to the adsorptive removal of cytokinesand there is no report about an adsorbent capable of improving theproliferative activity of lymphocytes. In recent years, the number ofpatients having activated lymphocyte therapy has been increasing year byyear with the marked advance of such therapy and, on the other hand, thenumber of patients relying on blood donation because of poor lymphocyteproliferation has also been increasing. In the case of blood donation,it is necessary to secure non-autologous plasma in conformity withpatient's therapeutic cycle. Further, there are a number of problems tobe taken up from the safety viewpoint, for example the risk ofinfection; therefore, it is desired that a method for overcoming thelymphocyte proliferation inhibition in lymphocyte culture by a simpleprocedure without losing other useful substances be developed.Furthermore, in cancer patients as well whose lymphocyte can proliferatein the presence of autologous body fluids, it is expected that furtherimprovements in proliferation rate and in cytokine producing activity,for instance, be achieved when the inhibition of lymphocyteproliferation in lymphocyte culture is broken down. Currently, however,neither adsorbent, nor porous body, nor device nor treatment method isavailable for such purposes. Further, adsorbents prepared by causing amaterial capable of adsorbing cytokines and like immunosuppressiveproteins to bind to a water-insoluble carrier have been disclosed(Patent Document 4 to 6). However, the effect of those adsorbentsdepends on an affinity between an amine residue and suchimmunosuppressive proteins as cytokines. Therefore, those adsorbentsrequire the presence of an amine residue.

At present, a treatment is eagerly anticipated which will promote theproliferation of those lymphocytes which are in a poorly proliferativecondition in lymphocyte culture for the treatment of a disease againstwhich a therapy comprising returning activated lymphocytes again intothe body, typically activated autologous lymphocyte therapy, istherapeutically effective.

Patent Document 1: Japanese Kokai Publication Sho-56-092824

Patent Document 2: Japanese Kokai Publication Hei-08-257398

Patent Document 3: Japanese Kokai Publication 2001-218840

Patent Document 4: Japanese Kokai Publication 2003-310751

Patent Document 5: Japanese Kokai Publication 2003-339854

Patent Document 6: Japanese Kokai Publication 2004-73618

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide anadsorbent useful in such treatment for promoting lymphocyteproliferation, a method for such treatment, and lymphocytes proliferatedby treatment with the porous material.

The present inventors made intensive investigations in search of aporous material capable of breaking down the inhibition of lymphocyteproliferation while minimizing the loss of useful substances necessaryfor the proliferation of lymphocytes, etc. and, as a result, found thatwhen a subject-derived body fluid is treated in advance with awater-insoluble porous material containing a high-molecular compoundhaving an angle of contact with water within the range of about 40° to98° or a porous material containing activated carbon, the lymphocyteproliferation rate in lymphocyte culture is markedly improved. Such andother findings have now led to completion of the present invention.

Thus, the present invention relates to

a porous material for body fluid treatment for promoting lymphocyteproliferation in lymphocyte culture

which contains a high-molecular compound having an angle of contact withwater within the range of 40° to 98°; and to

a porous material for body fluid treatment for promoting lymphocyteproliferation in lymphocyte culture

which comprises activated carbon.

The invention also relates to

a treatment device for promoting the lymphocyte proliferation inlymphocyte culture

which comprises the above-mentioned porous material and a containertherefor.

Further, the invention relates to

a method for proliferating lymphocytes;

a method for producing mammalian lymphocytes;

a method for producing a pharmaceutical composition;

an additive body fluid to be added to the culture medium on the occasionof lymphocyte culture;

a method for treating a disease against which returning extracorporeallyactivated mammalian lymphocytes into the body is therapeuticallyeffective; and

a use of activated carbon or a high-molecular compound having an angleof contact with water within the range of 40° to 98° in producing aporous material for body fluid treatment to promote the lymphocyteproliferation in lymphocyte culture.

DETAILED DESCRIPTION OF THE INVENTION

First, the porous material of the invention is described.

The porous material of the invention is a porous material for use inbody fluid treatment for promoting the lymphocyte proliferation inlymphocyte culture which contains a high-molecular compound having anangle of contact with water within the range of 40° to 98°.

In the practice of the invention, the angle of contact with water can bedetermined by preparing a smooth film specimen constituted of thehigh-molecular compound, which is the main constituent of the porousmaterial, forming a liquid drop on the film in a horizontal state usinga microinjector and measuring the contact angle at room temperature.When the porous material is soluble in an organic solvent, the contactangle can also be measured after dissolving the porous material andpreparing a cast film specimen on a flat sheet using the resultingsolution. For details of the measurement method, reference can be made,for example, to “Shin-Jikken Kagaku Koza (Lectures in ExperimentalChemistry, New Series) 18: Kaimen to Koroido (Interface and Colloid)”(First Edition, published October 20, Showa 52 (1977) by Maruzen Co.,Ltd.). Thus, a flat sheet specimen having a mirror-finish level ofsmoothness is placed horizontally so that the atmosphere surrounding thesame may be filled with the saturated vapor of the liquid to besubjected to measurement, and a liquid drop is formed thereon using amicroinjector. The size of the liquid drop is such that the contactdiameter is about 3 mm or smaller. The contact angle can be determinedby measuring the angle formed upon allowing the liquid drop to advancetoward the solid surface (at the time when after the liquid is allowedto develop and spread on the specimen, the liquid drop becomes stable ata certain size) using a reading microscope (having a magnification ofabout 20) equipped with a goniometer. The visibility of the imagebecomes very good when the lens barrel is inclined by 1 to 2 degreesdownward from the horizontal. The drop is illuminated from the frontwith light transmitted through an opalescent glass or with parallelbeams of light transmitted through a heat ray-absorbing glass.

The contact angle data reported herein were measured by the methoddescribed later in the example section.

The high-molecular compound having an angle of contact with water withinthe range of 40° to 98° includes as typical examples, but is not limitedto, synthetic high-molecular compounds such as nylon 6, nylon 6,6, nylon11, polyethylene, poly(vinylidene chloride), poly(vinyl chloride),poly(vinyl acetate), polystyrene, styrene-divinylbenzene copolymers,poly(trifluoroethylene), poly(chlorotrifluoroethylene), poly(ethyleneterephthalate), polypropylene, polyacrylic esters (e.g. poly(methylacrylate)), polymethacrylic esters (e.g. poly(methyl methacrylate)),crosslinked polyacrylates and crosslinked polyamides as well ascellulose and like water-insoluble ones. Among them, polymers orcopolymers produced by polymerizing an aromatic monomer or monomers(e.g. monomers selected from among alkylstyrenes which may optionally besubstituted, for example methylstyrene and ethylstyrene; divinylbenzeneand benzo-condensed cyclic compounds which may optionally besubstituted, for example divinylnaphthalene and divinylanthracene) arepreferred from the lymphocyte proliferation rate viewpoint. Inparticular, polystyrene and styrene-divinylbenzene copolymers arepreferred.

Any conventional polystyrene species can be used as the polystyrene. Thepolystyrene is an arbitrary styrene polymer or styrene-based polymer.

The styrene-divinylbenzene copolymers can be obtained by crosslinkingthe above-mentioned styrene compound with m-, o- or p-divinylbenzene,which may optionally be substituted.

The high-molecular compound mentioned above may optionally besubstituted by a halogen, alkyl, alkenyl, alkynyl, aralkyl, aryl,heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkylcarbonyl,alkoxycarbonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfonyl, etc.The substituents mentioned above may further be substituted by asubstituent(s) other than amines, and such substituents on theabove-mentioned substituents are preferably other than amide, urea,ester and ether groups.

The high-molecular compound mentioned above is preferably one having noamine residue bound thereto. As the amine residue, there may bementioned those residues resulting from chemical bonding of ammonia,primary to tertiary amine or the like to the high-molecular compound.

Further, the high-molecular compound mentioned above is preferably onecomprising no other compound immobilized thereon. The other compound isnot particularly restricted but includes, for example, amines, alcohols,glycidyl ethers, carboxylic acids and derivatives thereof, acid halides,halides, halogenated silanes, thiols, aldehydes and antibodies.

For the purpose of the present invention, namely from the lymphocyteproliferation promotion viewpoint, the above-mentioned angle of contactwith water is preferably greater than 60° but not greater than 96°, morepreferably about 70° to 94°, still more preferably about 75° to 91°.

In the practice of the invention, the porous material is preferablyinsoluble in water.

The porous material of the invention contains not less than about 50% byweight, preferably not less than about 60% by weight, more preferablynot less than about 70% by weight, still more preferably not less than80% by weight, of the high-molecular compound mentioned above.

As the component which can be contained in the porous material inaddition to the above-mentioned high-molecular compound having an angleof contact with water within the range of 40° to 98°, there may bementioned, for example, polyvinyl alcohol (contact angle 36°),poly(hydroxyethyl methacrylate) (contact angle 13°) and paraffin(contact angle 105 to 106°), among others.

By using two or more of the high-molecular compounds mentioned abovehaving an angle of contact with water within the range of 40° to 98° incombination and/or by using any of the above-mentioned high-molecularcompounds having an angle of contact with water of 40° to 98° as themain component and incorporating a component other than thehigh-molecular compound as the auxiliary material, among others, it ispossible to adjust the angle of contact with water of the porousmaterial to be obtained.

It is also preferred that the porous material have an angle of contactwith water within the range of the angle of contact with water of thehigh-molecular compound mentioned above. Thus, the angle of contact withwater of the porous material is preferably 40° to 98°, more preferablygreater than 60° but not greater than 96°, still more preferably about70° to 94°, particularly preferably about 75°to 91°.

The porous material of the invention occurs as a solid at ordinarytemperature and ordinary pressure and has pores appropriate in size,namely has a porous structure.

As for the size of pores in the porous material, the molecular-weightexclusion limit of the water-insoluble porous material as determinedusing polystyrene beads is preferably not higher than 1.5×10⁵, morepreferably not higher than 1.4×10⁵. When the molecular-weight exclusionlimit is higher than 1.5×10⁵, the level of unspecific adsorption tendsto increase, the loss of useful proteins in the body fluid tends tooccur and/or the ability of lymphocytes to proliferate tends to decreasewith ease. For lessening the influence of unspecific adsorption whilemaintaining the lymphocyte proliferation rate at high levels, themolecular-weight exclusion limit is more preferably not higher than1.3×10⁵, particularly preferably not higher than 1.2×10⁵, mostpreferably not higher than 1.0×10⁵.

The molecular-weight exclusion limit can be easily controlled, forexample, by adjusting the content of the above-mentioned high-molecularcompound, which is the main constituent of the porous material, in thestep of porous material production. Thus, as the content of thehigh-molecular compound increases, the molecular-weight exclusion limitlowers and, as the content of the high-molecular compound decreases, themolecular-weight exclusion limit rises.

The molecular-weight exclusion limit is the molecular weight of thosemolecules which cannot enter the pores (are excluded by the pores) inchromatography but are smallest in molecular weight among moleculesincapable of enter the pores.

The molecular-weight exclusion limit can be measured in the followingmanner. Polystyrene beads differing in particle diameter are passedthrough a column packed with the porous material, and the pore size isdetermined on the basis of an excluded polystyrene beads-based exclusioncurve. Then, the above-mentioned pore size is extrapolated to aspherical protein (e.g. dextran) the diameter and molecular weight ofwhich are known, to thereby determine the molecular weight on thespherical protein equivalent basis; this is regarded as themolecular-weight exclusion limit.

The shape of the porous material may effectively be spherical, granular,flat membrane-like, fibrous, or hollow fiber-like, for instance. Fromthe adsorption performance viewpoint, however, the spherical or granularform is more preferably used.

When the porous material is in a spherical or granular form, the averageparticle size thereof is preferably about 5 μm to 1,000 μm, morepreferably about 20 to 800 μm, still more preferably about 30 to 600 μm.

The average particle diameter can be determined in the following manner.The porous material in a wet state is developed on a dish and scores ofparticles are photographed using a CCD camera. Then, the averageparticle diameter is calculated from the image captured using theparticle diameter measurement software “Image-Pro plus” (product ofMedical Cybernetics, Inc.).

The porous material of the invention can be produced, for example, inthe following manner. One or more raw material monomer compounds aredispersed/suspended in a solvent having an appropriate viscosity (e.g.water). Suspension polymerization is carried out in the conventionalmanner while stirring the suspension to give the desired porousmaterial.

Further, the porous material of the invention may also compriseactivated carbon.

Usable as the activated carbon are, for example, fibrous activatedcarbon derived from phenolic fibers; coconut shell-derived activatedcarbon, petroleum pitch-derived activated carbon, peat-derived activatedcarbon, charcoal-based activated carbon and like granular activatedcarbon species.

The average particle diameter of the activated carbon is notparticularly restricted but is preferably about 5 μm to 1,000 μm, morepreferably about 20 to 800 μm, still more preferably about 30 to 600 82m.

Among the porous material components each comprising a high-molecularcompound or activated carbon, polystyrene, styrene-divinylbenzenecopolymers and activated carbon are preferred and, in particular,styrene-divinylbenzene copolymers are preferred, from the lymphocyteproliferation rate viewpoint.

As described hereinabove, activated carbon or a high-molecular compoundhaving an angle of contact with water within the range of 40° to 98° canbe used in producing the porous material for body fluid treatment forpromoting the lymphocyte proliferation in lymphocyte culture.

Now, the treatment device of the invention is a treatment device forpromoting lymphocyte proliferation in lymphocyte culture which comprisesthe porous material mentioned above as contained in a container.

The container to be used in the treatment device is not particularlyrestricted in shape, size or material.

The shape may be an arbitrary one, for example a sphere, container, bag,tube or column. As a preferred typical example, there may be mentioned,for example, a transparent or semitransparent cylindrical container witha capacity of about 0.1 to 400 ml and a diameter of about 0.1 to 10 cm.

The container can be manufactured using an arbitrary structure material.More specifically, as the structure material, mention may be made ofunreactive polymers, biocompatible metals, alloys and glass, forinstance.

As the unreactive polymers, there may be mentioned acrylonitrile-basedpolymers such as acrylonitrile-butadiene-styrene terpolymers;halogenated polymers such as polytetrafluoroethylene,polychlorotrifluoroethylene, tetrafluoroethylene-hexafluoropropylenecopolymers and polyvinyl chloride; polyamides, polysulfones,polycarbonates, polyethylene, polypropylene, polyvinyl chloride-acryliccopolymers, polycarbonate-acrylonitrile-butadiene-styrene, polystyreneand polymethylpentene, among others.

As metallic materials useful as the container material, there may bementioned stainless steel, titanium, platinum, tantalum, gold, andalloys thereof, as well as gold-plated alloy iron, platinum-plated alloyiron, cobalt-chromium alloys and titanium nitride-coated stainlesssteel, among others.

Materials resistant to autoclaving are particularly preferred and,specifically, silicone-coated glass, polypropylene, polyvinyl chloride,polycarbonates, polysulfones, polymethylpentene and the like may bementioned as such materials.

The treatment device is preferably one comprising a container having aliquid inlet and a liquid outlet and equipped with means for preventingporous material leakage and the water-insoluble porous material packedin the container, though the device is not limited to such one.

The means for preventing porous material leakage may be a mesh, nonwovenfabric, cotton plug or like filter.

The method for proliferating lymphocytes according to the invention isnow described.

The method for proliferating lymphocytes of the invention is a methodwhich comprises bringing the above-mentioned porous material intocontact with a body fluid and cultivating lymphocytes using the bodyfluid after contacting with the porous material.

As the method for proliferating lymphocytes of the invention, namely thetreating method for promoting lymphocyte proliferation, there mayspecifically be mentioned such methods as mentioned below. (1) Themethod which comprises packing the treatment device (container having abody fluid inlet and a body fluid outlet and equipped, at the outlet,with a filter allowing the body fluid passage but allowing no porousmaterial passage) with the porous material, bringing a body fluid intocontact therewith and then using the same in lymphocyte cultivation, (2)the method which comprises collecting a body fluid in a bag alreadycontaining the porous material and, after a predetermined period ofcontacting, filtering off the porous material and using the filtrate inlymphocyte cultivation, and (3) the method which comprises causing theporous material to coexist in the lymphocyte culture system andfiltering off the porous material from lymphocytes during cultivation orafter completion of cultivation.

Referring to the method (1), the method for contacting consists, forexample, in circulating the body fluid using a feed pump for a certainperiod of contacting or in allowing a certain period of contactingwithout circulation. As for the contacting time, 1 minute or a longerperiod of contacting is preferred and, from the adsorption performanceviewpoint, about 15 minutes to 6 hours of contacting is more preferred.From the sufficient adsorption performance and cell treatment efficiencyviewpoint, about 20 minutes to 4.5 hours of contacting is more preferredand about 30 minutes to 3 hours of contacting is still more preferred.

As the method (2), there are available, among others, the method whichcomprises collecting a body fluid directly in a bag already containingthe porous material and effecting a certain period of contacting; andthe method which comprises preparing a plasma or serum fraction fromblood by centrifugation, for instance, placing the fraction in such abag and effecting a certain period of contacting with the porousmaterial. As for the contacting time, about 1 minute or a longer periodof contacting is preferred and, from the adsorption performanceviewpoint, about 10 minutes to 10 hours of contacting is more preferredand about 15 minutes to 6 hours of contacting is still more preferred.From the sufficient adsorption performance and cell treatment efficiencyviewpoint, about 30 minutes to 3 hours of contacting is particularlypreferred. As another method, it is also possible to add the porousmaterial to a system wherein lymphocyte cultivation is in progress withpatient's plasma added, for causing the porous material coexist in thesystem and, after the lapse of a certain period of time, the porousmaterial is separated from lymphocytes by filtration using a filterallowing no passage of the porous material.

The temperature during contacting of the porous material with the bodyfluid in the above-mentioned method 1) or (2) can be arbitrarilyselected but is preferably about 4° C. to 50° C., more preferably about10° C. to 45° C.

Referring to the method (3), the porous material is caused to coexistwith lymphocytes in a lymphocyte culture vessel and the porous materialis separated from lymphocytes by filtration on the occasion of mediumexchange or after completion of the cultivation. The contacting time issuch that the contacting lasts until completion of lymphocytecultivation at the longest, and the amount of the porous material to beadded is preferably such that a sufficient space for lymphocyteproliferation can be secured without physical suppression thereof.

While the invention is not restricted to those mentioned above, theabove method (1) is procedurally simple and most preferred as the methodfor overcoming lymphocyte proliferation inhibition in lymphocyteculture.

There may be some unknown mechanism of inhibiting the lymphocyteproliferation, as referred to hereinabove. An unknown “lymphocyteproliferation inhibiting factor” may be involved in that mechanism.Therefore, the porous material of the invention can function as anadsorbent for lymphocyte proliferation inhibiting factors.

When the treatment device mentioned above is used in carrying out themethod for proliferating lymphocytes of the invention, an anticoagulantmay be used.

As the anticoagulant, use may be made of any of heparin, low-molecularheparin, nafamostat mesilate, gebexate mesilate, argatroban,acid-citrate-dextrose (ACD) solution, citrate-phosphate-dextrose (CPD)solution and like citrate-containing anticoagulants, among others. Amongthem, heparin may generally be mentioned as the most preferredanticoagulant.

In the case of serum preparation, for instance, the above-mentionedanticoagulant may not be contained in the serum.

The body fluid so referred to herein includes blood, plasma and serum.In addition, the body fluid includes other body fluids, such as asceticfluid, lymph and intraarticular fluid and fractions derived from theseas well as other living body-derived fluid components.

For the purpose of the invention, it is a simple and easy way to collectblood from the subject and, if desired, prepare plasma by separationfrom blood corpuscle fractions by such means as centrifugation, for thesubsequent use. Further, serum may be prepared for the subsequent use.

Also usable as the body fluid other than blood, plasma and serum aredilutions of these or supernatants obtained from them by pretreatment byspecific gravity gradient centrifugation using Ficoll, Percoll,Vacutainer tube, Lymphoprep or the like.

While the body fluid may be used immediately after blood collection,refrigerated or lyophilized blood and preparations may also be used.Further, it is also possible to treat the body fluid using the adsorbentand then refrigerate or lyophilize the same, followed by thawing for usewhen required. The invention is not restricted to such modes, however.

The body fluid released from lymphocyte proliferation inhibition inlymphocyte culture, after lymphocyte proliferation promoting treatmentaccording to the invention, can be obtained from the outlet side filterallowing no passage of the porous material as a result of filtering offof the porous material. When the target is blood, the desired plasma canbe obtained by further carrying out a centrifugation procedure. It isalso possible to obtain lymphocytes and the desired plasmasimultaneously from the blood after treatment with the porous materialby using Ficoll, Percoll, Vacutainer tube, Lymphoprep or the like.

The term “lymphocytes” as used herein refers to T cells and B cells,among others, occurring in mammalian peripheral blood, lymph vessels andbone marrow. The lymphocytes also include cells which are neither Tcells nor B cells, for example natural killer cells. The T cells are notparticularly restricted but include helper T cells, cytotoxic T cellsand killer T cells.

In the practice of the invention, the lymphocyte culture can be carriedout, for example, in the following manner. A mammalian body fluid isbrought into contact with the porous material mentioned above and thensowed in a lymphocyte culture medium. The lymphocyte culture medium isincubated at an arbitrary temperature (preferably about 20° C. to 45°C., more preferably about 30° C. to 40° C., still more preferably about37° C.) for an arbitrary period of time (for example about 3 days to 30days, preferably about 7 days to 21 days, more preferably about 10 daysto 18 days, most preferably about 14 days). As a result, lymphocytesproliferate.

The lymphocyte culture medium may be a conventional culture medium. Suchmedium includes, but is not limited to, such lymphocyte culture media asPB-MAX medium, AIM V medium, CHANG medium, LGM-3 medium, KBM400, GIT,Ham F-12, Dulbecco MEM, α-MEM, MEM, IMEM, RPMI-1640 and McCoy's 5Amedium, among others.

On the occasion of lymphocyte culture, an antibody or the like can alsobe used to activate lymphocytes. As the antibody, there may bementioned, for example, anti-CD3 antibody (OKT3) and so forth.

When the body fluid is a mammalian autologous body fluid and the mammalis in a condition of poor lymphocyte proliferation, the above-mentionedmethod for proliferating lymphocytes is more effective.

The term “poor lymphocyte proliferation” indicates that when patient'sbody fluid is added to a lymphocyte culture system, the lymphocyteproliferation rate is lower than the lymphocyte proliferation ratedetermined using a normal human-derived body fluid.

The lymphocyte proliferation rate is the proliferation rate (number ofcells after 7 days/number of cells sowed) found after 1 week oflymphocytes cultivation at 37° C. using a culture medium supplementedwith 0.1% (v/v) to 20% (v/v) of a patient-derived body fluid treatedwith the porous material or a normal human-derived body fluid not yettreated with the porous material.

In the case of packing a column with the porous material for using thesame, it is important that clogging will never occur on the occasion offluid passage, among others. For that reason, the porous material isrequired to have sufficient mechanical strength. Therefore, the porousmaterial to be used in the practice of the invention is more preferablya hard one. In the case of a granular gel material, the term “hard one”as used herein refers to a gel which, when a cylindrical column isuniformly packed with the gel and an aqueous fluid is passedtherethrough, shows a linear relationship between pressure loss ΔP andflow rate until about 0.3 kg/cm². In the case of using the porousmaterial by placing the same in a bag, however, it may be a soft one.

Now, the method for producing mammalian lymphocytes according to theinvention is a method which comprises bringing a mammalian body fluidinto contact with the porous material mentioned above, cultivatinglymphocytes using the body fluid after contacting with the porousmaterial, and recovering lymphocytes produced.

The lymphocyte recovery can be carried out by any arbitrary method, forexample by centrifugation, membrane filtration or chromatography.

Now, the method for manufacturing a pharmaceutical composition accordingto the invention is a method which comprises producing lymphocyte by theabove-mentioned lymphocyte production method and blending thelymphocytes with a pharmaceutically acceptable additive.

As the pharmaceutically acceptable additive, there may be mentioned, forexample, anticoagulants, vitamins and other nutrient sources, andantibiotics.

The pharmaceutical composition manufacture can be carried out using anacceptable pharmaceutical technique to give an appropriate galenic formcomposition (e.g. for transfusion, drip infusion or injection).

The additive body fluid of the invention, which is to be added to aculture medium on the occasion of lymphocyte culture, is one obtained bybringing a mammalian body fluid into contact with the porous materialmentioned above.

The additive body fluid can be prepared, for example, by bringing a bodyfluid derived from a mammal with or without a disease against which atherapeutic effect is produced by extracorporeally activatinglymphocytes and returning them into the body into contact with theporous material mentioned above.

Further, the method of the invention for treating a disease againstwhich a therapeutic effect is produced by extracorporeally activatinglymphocytes and returning them into the body is a method which comprisesbringing a body fluid derived from a mammal requiring or not requiringtreatment into contact with the porous material mentioned above,cultivating lymphocytes using the body fluid after contacting with theporous material, and administering the thus-obtained lymphocytes to themammal.

The disease against which a therapeutic effect is produced byextracorporeally activating lymphocytes and returning them into the bodyincludes, but is not limited to, cancer, infectious diseases and immunediseases, among others.

The activation includes, within the meaning thereof, an increase in thenumber of lymphocytes, a change in lymphocyte population as a result ofproliferation and/or an improvement in the functions intrinsic inlymphocytes, and the like.

(Effects of the invention)

The present invention has made it possible to overcome the lymphocyteproliferation inhibition in lymphocyte culture and thus markedlyincrease the lymphocyte proliferation rate by adsorbing lymphocyteproliferation inhibiting factors from a body fluid derived from asubject with a disease against which a therapeutic effect is produced byextracorporeally activating lymphocytes and returning them into thebody, for example a cancer subject with poor lymphocyte proliferation,without adsorbing factors necessary for lymphocyte proliferation fromthat body fluid. The invention is useful in that it provides a porousmaterial for relieving the lymphocyte proliferation inhibition inlymphocyte culture using a target body fluid in activated autologouslymphocyte therapy, for instance, according to which a disease istreated or the progress of a disease is inhibited by takingimmunocompetent cells (lymphocytes in particular) in blood out of thebody, culturing them for stimulation/activation and for proliferationand again returning them into the body, as well as a method forproliferating lymphocytes which utilizes the above-mentioned porousmaterial.

BEST MODES FOR CARRYING OUT THE INVENTION

The following examples specifically illustrate the present invention.These examples are, however, by no means limitative of the scope of theinvention.

In the following examples etc., the angle of contact with water,molecular-weight exclusion limit and average particle diametermeasurements were made in the following manner.

(1) Angle of Contact with Water

A flat and smooth film was prepared by compressing the high-molecularcompound sample at a high pressure. The thus-obtained flat and smoothsheet specimen was placed horizontally, and a liquid drop was formedthereon using a microinjector. The size of the liquid drop was such thatthe contact diameter was about 1 to 2 mm. The contact angle wasdetermined by measuring the angle formed upon allowing the liquid dropto advance toward the solid surface, at room temperature (20° C.), usinga reading microscope (having a magnification of about 20) equipped witha goniometer.

(2) Molecular-weight Exclusion Limit

Polystyrene beads differing in particle diameter were passed through acolumn packed with the porous material, and the pore size was determinedon the basis of an excluded polystyrene beads-based exclusion curve.Then, the above-mentioned pore size was extrapolated to the sphericalprotein dextran the diameter and molecular weight of which were known,to thereby determine the molecular weight on the dextran equivalentbasis; this was reported as the molecular-weight exclusion limit.

(3) Average Particle Diameter

The porous material in a wet state was developed on a dish and scores ofparticles were photographed using a CCD camera. Then, the averageparticle diameter was calculated from the image captured using theparticle diameter measurement software “Image-Pro plus” (product ofMedical Cybernetics, Inc.).

EXAMPLE 1 (1) Lymphocyte Preparation

A winged needle for intravenous injection was connected to an adaptorand a holder was connected to the other end of the adaptor. Theinjection needle was stabbed into the brachial region of a normalsubject and about 7.5 ml of blood was collected in a tube for lymphocyteseparation (Vacutainer tube (product of Becton Dickinson and Company)).After blood collection, the Vacutainer tube was subjected to 20 minutesof centrifugation at 3,000 rpm at room temperature. The lymphocyte layerwas recovered and supplemented with 40 ml of physiological saline, andthe resulting mixture was centrifuged at 1,500 rpm at 4° C. for 5minutes. This procedure was repeated several times for washinglymphocytes, and a lymphocyte suspension with a predeterminedconcentration was prepared by resuspending the lymphocytes in KBM 400medium (product of Kohjin Bio Co., Ltd.).

(2) Preparation of a Plate with OKT3 Immobilized Thereon

OKT3(product of Dainippon Pharmaceutical) was diluted to a concentrationof 5 μg/ml with physiological saline, and the dilution was distributedin 500-μl portions into the wells of a 24-well polystyrene microplate(product of Sumitomo Bakelite Co., Ltd.). After 2 hours of standingstill at room temperature, the OKT3 solution was removed, and the platewas washed with two equal portions of physiological saline; a plate withOKT3 immobilized thereon was thus prepared.

(3) Preparation of a Porous Material

A monomer mixture composed of 100 parts by weight of divinylbenzene forindustrial use (divinylbenzene content 57%), 100 parts by weight oftoluene, 60 parts by weight of isoamyl alcohol and 1 part by weight ofbenzoyl peroxide (content 75%) was added to an aqueous solution composedof 572 parts by weight of water, 23 parts by weight of sodium chloride,1 part by weight of polyvinyl alcohol and 0.03 part by weight of sodiumnitrite, and the polymerization was carried out at 80° C. in a nitrogenatmosphere for 5 hours with stirring so that droplets of the monomermixture might be dispersed and suspended. The polymer particles formedwere filtered off, washed with water and then deprived of such residualcomponents as the solvent, monomer and initiator by extraction withacetone and again thoroughly washed with water and hot water. Porousstyrene-divinylbenzene copolymer beads with a volume average particlediameter of about 400 μm were obtained (molecular-weight exclusion limitabout 8×10⁴, angle of contact with water about 85°).

(4) Plasma Treatment

The porous styrene-divinylbenzene copolymer beads were thoroughly washedwith physiological saline and then 0.17 ml thereof were measured andplaced in a cryotube. The physiological saline was thoroughly removedfrom the porous beads, 1 ml of cancer patient's serum was added thereto,followed by 2 hours of incubation at 37° C. with stirring (40 rpm) on aMIX rotor.

(5) Lymphocyte Cultivation

The plasma treated in the above manner was added to the lymphocyteculture medium KBM 400 (product of Kohjin Bio Co., Ltd.) to aconcentration of 9% (v/v). Using the resulting culture medium, alymphocyte suspension with a lymphocyte number of 1.0×10⁵ cells/ml wasprepared and sowed onto the previously prepared plate with OKT3immobilized thereon in an amount of 444 μl/well (n=3 wells).

After 7 days of cultivation, lymphocytes were recovered and the numberof cells was counted using a hemocytometer, and the lymphocyteproliferation rate was calculated according to the following formula(1).

Proliferation rate (times)=number of lymphocytes after 7 days ofcultivation/number of lymphocytes sowed  (1)

As a result, in the case of treatment of cancer patient's plasma withthe adsorbent, the number of lymphocytes increased to 16.7 times(proliferation rate) the number of cells sowed.

EXAMPLE 2

The lymphocyte proliferation rate was determined in the same manner asin Example 1 except that petroleum pitch-derived activated carbon with aparticle diameter of about 500 μm was used as the adsorbent in lieu ofthe porous styrene-divinylbenzene copolymer beads. As a result, thenumber of lymphocytes increased to 10.6 times (proliferation rate) thenumber of cells sowed.

EXAMPLE 3

The lymphocyte proliferation rate was determined in the same manner asin Example 1 except that porous polystyrene (angle of contact with waterabout 85° C.) beads with a molecular weight exclusion limit of nothigher than 1×10⁴ and a particle diameter of about 400 μm were used. Asa result, the number of lymphocytes increased to 11.5 times(proliferation rate) the number of cells sowed.

EXAMPLE 4

Porous material preparation: A cellulose solution with a viscosity ofabout 1,000 cP was jetted, in the form of uniform droplets, into agaseous phase under direct application of vibrations at a frequency ofabout 20,000 Hz to the solution. After causing the droplets to make asufficient flight to take a spherical form, they were captured in acoagulation bath, deprived of the solvent and washed to give porouscellulose particles with a particle diameter of about 400 μm(molecular-weight exclusion limit not higher than 3×10⁴, angle ofcontact with water about 50°).

The lymphocyte proliferation rate was determined in the same manner asin Example 1 except that the porous particles were used. As a result,the number of lymphocytes increased to 6.9 times (proliferation rate)the number of cells sowed.

EXAMPLE 5

The lymphocyte proliferation rate was determined in the same manner asin Example 1 except that porous cellulose beads with a molecular-weightexclusion limit of not higher than 6×10⁴ and a particle diameter ofabout 400 μm (angle of contact with water about 40°) were used. As aresult, the number of lymphocytes increased to 5.7 times (proliferationrate) the number of cells sowed.

COMPARATIVE EXAMPLE 1

The lymphocyte proliferation rate was determined in the same manner asin Example 1 except that the patient's plasma was used withoutcontacting with any adsorbent. As a result, the number of lymphocytesincreases to 3.9 times (proliferation rate) the number of cells sowed.

COMPARATIVE EXAMPLE 2

The lymphocyte proliferation rate was determined in the same manner asin Example 1except that the porous particles used in Example 4 were usedafter binding dextran sulfate thereto via epichlorohydrin(molecular-weight exclusion limit not higher than 3×10⁴, angle ofcontact with water about 35°). As a result, the number of lymphocytesincreased to 3.6 times (proliferation rate) the number of cells sowed.

COMPARATIVE EXAMPLE 3

The lymphocyte proliferation rate was determined in the same manner asin Example 1 except that the porous particles used in Example 5 wereused after binding dextran sulfate thereto via epichlorohydrin(molecular-weight exclusion limit not higher than 3×10⁴, angle ofcontact with water about 30°). As a result, the number of lymphocytesincreased to 3.2 times (proliferation rate) the number of cells sowed.

The results of the lymphocyte proliferation rate evaluation in Examples1 to 5 and Comparative Examples 1 to 3 are shown in Table 1. From theseresults, it is evident that the lymphocyte proliferation rate could bemarkedly increased by cultivating lymphocytes using the specific porousmaterials of the invention after contacting with a body fluid ascompared with the case of non-use of the porous material (adsorbent) orthe cases where dextran sulfate was immobilized on the high-molecularcompounds to render them hydrophilic.

TABLE 1 Influence of use of patient's plasma on lymphocyte proliferationLymphocyte proliferation rate after 7 days of cultivation (calculatedaccording to equation 1)) Lymphocyte proliferation rate (times) Example1 16.7 ± 3.2  Example 2 10.6 ± 2.7  Example 3 11.5 ± 3.0  Example 4 6.9± 1.5 Example 5 5.7 ± 1.6 Comp. Ex. 1 3.9 ± 1.4 Comp. Ex. 2 3.6 ± 0.8Comp. Ex. 3 3.2 ± 0.5 n = 3 ± S.D

INDUSTRIAL APPLICABILITY

The present invention has made it possible to overcome the lymphocyteproliferation inhibition in lymphocyte culture and thus markedlyincrease the lymphocyte proliferation rate by adsorbing lymphocyteproliferation inhibiting factors from a body fluid derived from asubject with a disease against which a therapeutic effect is produced byextracorporeally activating lymphocytes and returning them into thebody, for example a cancer subject with poor lymphocyte proliferation,without adsorbing factors necessary for lymphocyte proliferation fromthat body fluid. The invention is useful in that it provides a porousmaterial for relieving the lymphocyte proliferation inhibition inlymphocyte culture using a target body fluid in activated autologouslymphocyte therapy, for instance, according to which a disease istreated or the progress of a disease is inhibited by takingimmunocompetent cells (lymphocytes in particular) in blood out of thebody, culturing them for stimulation/activation and for proliferationand again returning them into the body, as well as a method forproliferating lymphocytes which utilizes the above-mentioned porousmaterial.

1. A porous material for body fluid treatment for promoting lymphocyteproliferation in lymphocyte culture which contains a high-molecularcompound having an angle of contact with water within the range of 40°to 98°.
 2. The porous material according to claim 1 which has amolecular-weight exclusion limit of not higher than 1.5×10⁵.
 3. Theporous material according to claim 1, wherein the high-molecularcompound is an aromatic high-molecular compound.
 4. The porous materialaccording to claim 3, wherein the aromatic high-molecular compound ispolystyrene or a styrene-divinylbenzene copolymer.
 5. The porousmaterial according to claim 1, wherein the high-molecular compoundcontains no amine residue bound thereto.
 6. The porous materialaccording to claim 1, wherein the high-molecular compound has no othercompound immobilized thereon.
 7. A porous material for body fluidtreatment for promoting lymphocyte proliferation in lymphocyte culturewhich comprises activated carbon.
 8. A treatment device for promotinglymphocyte proliferation in lymphocyte culture which comprises theporous material according to claim 1 and a container therefor.
 9. Thedevice according to claim 8, wherein said container has a liquid inletand a liquid outlet.
 10. A method for proliferating lymphocytes whichcomprises bringing a body fluid into contact with the porous materialaccording to claim 1 and cultivating lymphocytes using the body fluidafter contacting with said porous material.
 11. The method forproliferating lymphocytes according to claim 10, wherein said body fluidis an autologous body fluid of a mammal and said mammal is in acondition of poor lymphocyte proliferation.
 12. A method for producingmammalian lymphocytes which comprises bringing a mammalian body fluidinto contact with the porous material according to claim 1, cultivatinglymphocytes using the body fluid after contacting with the porousmaterial and recovering the thus-produced lymphocytes.
 13. A method forproducing a pharmaceutical composition which comprises producinglymphocytes according to the method according to claim 12 and blendingthe lymphocytes with a pharmaceutically acceptable additive.
 14. Anadditive body fluid to be added to the culture medium on the occasion oflymphocyte culture which is obtained by bringing a mammalian body fluidinto contact with the porous material according to claim
 1. 15. A methodfor treating a disease against which a therapeutic effect is obtained byreturning extracorporeally activated mammalian lymphocytes into the bodywhich comprises bringing a body fluid derived from a mammal requiring ornot requiring treatment into contact with the porous material accordingto claim 1, cultivating lymphocytes using the body fluid aftercontacting with the porous material, and administering the thus-obtainedlymphocytes to said mammal.
 16. The use of activated carbon or ahigh-molecular compound having an angle of contact with water within therange of 40° to 98° in producing a porous material for body fluidtreatment to promote the lymphocyte proliferation in lymphocyte culture.17. The porous material according to claim 2, wherein the high-molecularcompound is an aromatic high-molecular compound.
 18. The porous materialaccording to claim 2 wherein the high-molecular compound contains noamine residue bound thereto.
 19. The porous material according to claim3 wherein the high-molecular compound contains no amine residue boundthereto.
 20. The porous material according to claim 4 wherein thehigh-molecular compound contains no amine residue bound thereto.